As a Visiting Scientist working on detail from NINDS IRP at the VA/UMaryland Research facility, my research efforts cover a wide area due to multiple collaborations, which were chosen as they address elements of the goal to deliver potentially therapeutic genes to the CNS in order to combat neurodegenerative diseases. A third generation lentiviral vector is at the center of this effort, because it can transduce non-dividing and dividing cells like stem cells, and integrate and stably express the encoded transgene. Depending on the target tissue and on in vivo studies, at a later time, it may be necessary for efficacy to create different vector pseudotypes or to use one of the adeno-associated virus serotypes. All of the lentiviral vector DNA constructs listed below were completed at the VA/UMaryland or have previously been assembled in my lab at NINDS. In most cases, small amounts of vector have been isolated, which can now be tested for transgene function. The vector constructs encode the following transgenes: 1. Transgene encoding a transneuronal proteins and less invasive routes of gene delivery to the CNS: The EGFP-TTC (a EGFP-tetanus toxin C fragment fusion protein) serves as a model protein for transneuronal transport and dissemination to connected neurons. The transgene will be delivered using lentiviral vector pseudotyped with either rabies virus glycoprotein G or vesicular stomatitis virus G via intramuscular injection or via nasal instillation. We demonstrated earlier that muscle fibers and motor neurons are efficiently transduced using a lentiviral vector pseudotyped with select rabies virus G proteins. The olfactory epithelium with its neuronal connections and its potential delivery route for the treatment of neurodegenerative diseases like Alzheimers disease, is a low invasive site for CNS entry. The olfactory epithelium contains dividing precursor cells, and transductions by a lentiviral vector would allow to continuously express the transgene. This is also the case with motor neurons after retrograde transgene delivery of the vector after infection at neuromuscular junction. Transduction and expression of the EGFPTTC gene in myocytes, motor neurons and olfactory epithelial cells will be evaluated and subsequently the transneuronal transport of EGFP-TTC protein across synapses of connected neurons. The extent of this transport and the presence of EGFP-TTC in higher order neurons will be important. It may be indicative for the future delivery and dissemination of potentially therapeutic proteins to larger regions of the brain, proteins that originate from fewer transduced cells and are delivered with low invasiveness. 2. Transgenes for neuroprotection/restoration: a) The transcription factor Nrf2 and a constitutively active form of Nrf2, both expressed from a CMV or an astrocyte-specific promoter in primary cells, or potentially expressed in neural stem cells, may increase the resistance of these cells to toxic insults after transplantation. Importantly, increased Nrf2 expression in astrocytes can protect neighboring neurons via a by-stander effect. b) Transgenes to augment mitochondrial function to combat oxidative stress during peripheral neuropathy in diabetic patients: PGC1a, TFAM and Sirt1. c) IL-10 to prevent migroglia infiltration, which has been identified as a contributing factor causing chronic pain. d) Smn1 for gene replacement in spinal muscular atrophy in motor neurons. An answer to the question, whether the delivery of the Smn1 gene to motoneurons can restore the normal phenotype in animal models of SMA models at any time after birth, as compared to a limited time window during development requiring the presence of Smn1, has important implications for any therapeutic strategy of spinal muscular atrophy. 3. a) Transgenes that may stimulate neuronal development (dopaminergic neuron) of human fetal neural progenitor cells: the transcription factors NeuroD2 and PitX3. If successful these cells may be a source of new neurons. b) CD34 subtype as neural precursor cells (?): Vectors encoding three cell-type specific promoters expressing different color reporter transgenes: Syn-DsRed2 (neurons), GFAP-ECFP (astrocytes), MBP-EYFP (oligodendrocytes). CD34 hematopoietic stem cell subtype will be transduced with these vectors, expanded in vitro and injected into the tail vein of mice. The migration of these cells across the blood-brain-barrier into the CNS and the cell type-specific activation of these 3 promoters would provide strong evidence that this subtype of CD34 cells can indeed differentiate into the 3 neural cell types. Depending on the extent of migration and differentiation, these precursor cells may open another possibility for less invasive gene delivery to the CNS. These cells normally differentiate into microglia and migrate into the CNS to sites of inflammation. Thus, gene modified CD34 cells may be useful for example in multiple sclerosis.